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      Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation

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          Abstract

          Background

          Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)].

          Methods

          Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC–MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes.

          Results

          Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression.

          Conclusions

          Nasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12967-016-0927-4) contains supplementary material, which is available to authorized users.

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          Most cited references35

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          Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.

          Atopic disease, including atopic dermatitis (eczema), allergy and asthma, has increased in frequency in recent decades and now affects approximately 20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable. Although most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic variants (R510X and 2282del4) in the gene encoding filaggrin (FLG) are very strong predisposing factors for atopic dermatitis. These variants are carried by approximately 9% of people of European origin. These variants also show highly significant association with asthma occurring in the context of atopic dermatitis. This work establishes a key role for impaired skin barrier function in the development of atopic disease.
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            Large-scale proteomics and phosphoproteomics of urinary exosomes.

            Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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              Exosomes with immune modulatory features are present in human breast milk.

              Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30-100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant's immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-gamma production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3(+)CD4(+)CD25(+) T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.
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                Author and article information

                Contributors
                +46 031 786 67 11 , cecilia.lasser@gu.se
                serena.oneil@gu.se
                ganesh.skelke@gu.se
                carina.sihlbom@gu.se
                sara.hansson@astrazeneca.com
                ysgho@postech.ac.kr
                bo.lundback@gu.se
                jan.lotvall@gu.se
                Journal
                J Transl Med
                J Transl Med
                Journal of Translational Medicine
                BioMed Central (London )
                1479-5876
                20 June 2016
                20 June 2016
                2016
                : 14
                : 181
                Affiliations
                [ ]Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
                [ ]Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
                [ ]Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea
                Article
                927
                10.1186/s12967-016-0927-4
                4913423
                27320496
                f619c4c3-dda0-4b95-a3cd-14ae996cee79
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 9 October 2015
                : 30 May 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet (SE);
                Award ID: K2011-56X-20676-04-6
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004722, Stiftelsen Lars Hiertas Minne (SE);
                Funded by: VBG Group Herman Krefting Foundation for Asthma and Allergy Research
                Funded by: FundRef http://dx.doi.org/10.13039/501100003793, Hjärt-Lungfonden;
                Award ID: 20120528
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100002794, Cancerfonden (SE);
                Award ID: CAN2012/690
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Medicine
                asthma,chronic rhinosinusitis,exclusion list,exosomes,extracellular vesicles,cell migration,mass spectrometry,nasal lavage fluid,proteomics,tandem mass tags

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